FLIM - Fluorescence Lifetime Imaging Microscopy

Fluorescence lifetime imaging microscopy (FLIM) is a technique utilized to measure the fluorescence lifetime of molecules. The fluorescence lifetime is the average time a molecule spends in an excited state before returning to the ground state. This measurement is a highly quantitative value that can be used to determine molecular dynamics with nanoscale resolution. FLIM is a standard technique for determining the lifetimes of fluorescent molecules in cells, tissues, and whole animal models.

A key advantage of fluorescence lifetime is that it does not change with variations in local fluorophore concentration and is independent of fluorescence concentration, excitation, and photobleaching. The lifetime of the molecule, however, is dependent on changes in its micro-surroundings and conformation state. These can be introduced via small changes in ion concentration, pH, lipophilicity, and even interactions with other molecules. Changes in its micro-environment or interactions will cause the excited fluorescent molecule to lose its energy faster, resulting in a decrease in lifetime.

There are two primary implementations of FLIM: (1) time-domain FLIM, which uses a pulsed light source in conjunction with either time-gated (i.e., wide-field) or time-correlated (i.e., single-point) detection, and (2) frequency-domain FLIM, which utilizes a sinusoidally modulated light source with either a sinusoidally modulated, intensified CCD detector to achieve wide-field imaging or a modulated single-element detector to perform scanning or single-point detection.

PI’s Picks

For conducting time-resolved FLIM, Princeton Instruments recommends the PI-MAX4:1024i, an ICCD camera platform that fiberoptically bonds any one of several conventional intensifiers to the CCD and uses state-of-the-art electronics to gate the intensifier (which normally achieves ~2 to 3 nsec gating) at <500 psec without sacrificing quantum efficiency. An oscilloscope-like LightField user interface delivers complete experiment control and the camera can be operated remotely via a GigE data interface.

For performing frequency-domain FLIM as well as time-resolved FLIM, the PI-MAX4:1024i-RF is recommended. By modulating the gain of its fiberoptically coupled, high-sensitivity Gen III filmless intensifier at a radio frequency (RF) rate using the camera system’s own internal electronics, this ICCD camera operates as a 2D lock-in amplifier. It features two independent, built-in, direct digital synthesizers: one generates the RF to modulate the intensifier (1–200 MHz) and the other provides a user-controlled RF signal to modulate the illumination so as to accomplish RF phase-sensitive detection. LightField software lets users select modulation frequency, control phase sweep range and granularity (in 1 degree steps, up to 360 degrees), and set RF output p-p voltage levels. GigE interface supported.

The system setup for RLD-facilitated TRFLIM employs a pulsed laser and an optical microscope, as well as a gated ICCD camera from Princeton Instruments. (Diagram courtesy of Dr. Ammasi Periasamy, W.M. Keck Center for Cellular Imaging, University of Virginia.) More details, along with images and data, can be found in “Novel Time-Resolved FLIM Measurements Method”.

An advanced, frequency-domain FLIM experiment:

A typical experimental setup for frequency-domain homodyne FLIM uses a modulated image intensifier and CCD detector. Diagram and photo courtesy of Annette Buntz (Zumbusch Group, University of Konstanz, Germany). More details, along with images and data, can be found in “Wide-Field, Frequency-Domain FLIM Measurements Made Simple”.

Application Notes

Wide-Field, Frequency-Domain FLIM Measurements Made Simple
Learn more about how this application was enabled by the PI-MAX 4 1024i RF ICCD camera, allowing researchers to perform these wide-field measurements with unprecedented ease in terms of intensifier modulation and instrument synchronization, as well as with minimal external equipment.

Tech Notes

emICCD: The Ultimate in Scientific ICCD Technology
With the rapid expansion of research in areas such as nanotechnology, quantum computing, and combustion, the development of higher-performance time-gated cameras is becoming a necessity. This technical note describes the latest breakthrough in scientific intensified CCD (ICCD) technology: the world’s first emICCD.